Stimulation of the T cell antigen receptor (TCR) by antigen or antibodies initiates signal transduction events that lead to T cell responses. Critical for TCR signal transduction is the phosphorylation of a number of proteins. Among these is the proto-oncogene Vav1 which plays a critical role in T and B cell development and in antigen receptor-mediated signal transduction. The precise function of Vav1 is not known, but it has been implicated in signal transduction events leading to cytoskeletal and transcriptional regulation. The Vav family of proteins contains multiple modular domains including a guanine nucleotide exchange factor (GEF) domain that can regulate small GTPases of the Rho family. Tyrosine phosphorylation of Vav1 regulates its GEF function. Rho GTPases regulate actin cytoskeleton reorganization, which could contribute to TCR signaling, perhaps by forming a matrix for the nucleation of signaling machinery at the site of TCR engagement. We have made considerable progress regarding Vav1 function in the previous funding cycle. Our studies of Vav1 phosphorylation have revealed that Src and Syk kinases both play a role. We identified a negative regulatory site, Y174, in Vav1. Mutation of Y174 activated Vav1 GEF activity and its effects on TCR-induced transcriptional responses, but the mechanism is not clear. It is likely to involve phosphorylation of Y174. Since the Vav 1-mediated activation of Rho GTPases might regulate TCR signaling function, we focused on one of their downstream effectors, the serine/threonine kinase Pak1. We found that Pak1 is activated by TCR stimulation and that it contributes to TCR-induced transcriptional responses. Epistasis experiments suggest that Pak1 function is downstream of Vav1 but upstream of Ras and calcium responses. Recent studies in our laboratory have also implicated another GEF domain-containing protein, PIX, in the regulation of Pak1. The relative contribution of Vav1 and PIX towards Pak1 activation and actin cytoskeletal reorganization is not clear. Surprisingly, our studies of Y174 also led us to identify a GEF-independent function of Vav1 that can contribute to TCR signals leading to transcriptional responses. Since Vav proteins contain multiple protein interaction modules, it is not surprising that at least one other domain contributes to Vav1 effects on transcription. In efforts to further characterize the function of Vav1 and its interacting proteins, we focused on SLP-76 which inducibly interacts with Vav1 via the Vav1 SH2 domain following TCR stimulation. We isolated a Jurkat SLP-76 deficient mutant that helped us to define some of the TCR-mediated signaling events that require SLP-76, optimal Ras and PLCy1 activation. The proposed studies in this application will build on these observations to further explore the mechanism by which Vav1 contributes to TCR signaling. Our specific aims are: 1) to understand the mechanism by which Y174 negatively regulates Vav1 function; 2) to define the relative roles of Vav1 and PIX in activating Pak1 and understand how Pak regulates TCR signal transduction pathways; 3) to identify the domain(s) and mechanism(s) responsible for the GEF-independent Vav1 function that regulates NFAT-regulated transcriptional responses; and 4) To define the mechanism for LAT-dependent Rac activation. These studies will provide a comprehensive view of how Vav1 and Pak1 are regulated and contribute to TCR signaling.